The present invention relates to the selective cleavage of a target nucleic acid using a cleavage compound. Inhibition of messenger RNA translation can be achieved using an anti-sense oligonucleoside conjugated to a cleavage-enhancing metal complex, which hybridizes to the target nucleic acid to effect cleavage at a target site in the 5xe2x80x2-cap structure of the nucleic acid.
The possibility of developing therapeutic agents which bind to critical regions of RNA, for example mRNA, and selectively inhibit the function, replication or survival of abnormal cells or foreign organisms is an exciting concept. See, e.g., Dervan, Science 1988; 232:464-471. Various laboratories have pursued the design and development of molecules which interact with DNA in a sequence-specific manner. Such molecules have been proposed to have far-reaching implications for the diagnosis and treatment of diseases involving foreign genetic materials (such as viruses) or alterations to genomic DNA (such as cancer).
Anti-sense oligonucleotides are one type of sequence-specific molecule that has been demonstrated to be effective for inhibition of virus and human genes. In one application of this technology, anti-sense oligonucleotides are complementary to at least a portion of the messenger RNA (MRNA) transcribed from the target gene and can hybridize with the mRNA, thereby preventing ribosomal translation and subsequent protein synthesis. Anti-sense oligonucleotides have been shown to mediate inhibition of the Rous Sarcoma virus in tissue cultures (Zamecnik and Stephenson, Proc. Natl. Sci. U.S.A. 1978; 75:280-284) as well as the HTLV-III (HIV-1) virus (Zamecnik, et al., Proc. Natl. Acad. Sci. U.S.A. 1986; 83:4145-4146). Anti-sense oligonucleotides also have been shown to suppress the expression of selected non-viral genes in vitro, such as rabbit-globin (Goodchild, et al., Arch. Biochem. Biophys. 1988; 264:401-409) and human c-myb (Anfossi, et al., Proc. Natl. Acad. Sci. U.S.A. 1989; 86:3379).
Naturally-occurring oligonucleotides are subject to degradation or inactivation by cellular endogenous nucleases. Since anti-sense oligonucleotides must remain intact to be effective, some researchers have modified oligonucleotides to make them resistant to degradation or inactivation by nucleases. These modified oligonucleotides typically contain altered internucleoside linkages in which one or more of the naturally occurring phosphodiester linkages has been replaced. Oligonucleosides having phosphoroamidate or phosphorothioate linkages have been shown to increase the inhibition of HIV-1 in tissue cultures. See, e.g. Agarwal, et al., Proc. Natl. Acad. Sci. U.S.A. 1988; 85:7079-7083.
Nuclease-resistant nonionic oligonucleosides having methylphosphonate linkages have also been studied in vitro and in vivo as potential anticancer, antiviral and antibacterial agents. Miller, et al., Anti-Cancer Drug Design, 2:117-128 (1987). For example, anti-sense oligonucleosides containing methylphosphonate linkages have been demonstrated to inhibit HIV-induced syncytium formation. Sarin, et al., Proc. Natl. Acad. Sci. U.S.A. 1988; 85:7448-7451. The internucleoside bonds of these analogs are said to approximate the conformation of phosphodiester bonds in nucleic acids. It has been noted that the nucleic acid phosphate backbone in a methylphosphonate linkage is rendered neutral by methyl substitution of one anionic phosphoryl oxygen. This substitution is thought to decrease inter- and intra-strand repulsion attributable to charged phosphate groups. Miller, et al., Anti-Cancer Drug Design 2:117-128 (1987).
Oligonucleotide analogs with a methylphosphonate backbone are believed to be capable of penetrating living cells and have been reported to inhibit mRNA translation in globin synthesis and vesicular stomatitis viral protein synthesis and to inhibit herpes simplex virus replication by preventing splicing of pre-mRNA. Blake, et al., Biochemistry 1985; 24:6132-6138; Blake, et al., Biochemistry 1985; 24:6139-6145; Murakami, et al., Biochemistry 1985; 24:40414046; Miller, et al., Biochimie 1985; 67:769-776; Agris, et al., Biochemistry 1986; 23:6268-6275. Mechanisms of action for inhibition by the methylphosphonate analogs include formation of stable complexes with RNA and/or DNA having a substantially complementary nucleic acid sequence.
Nonionic oligonucleotide alkyl- and aryl-phosphonate analogs complementary to a selected single stranded foreign nucleic acid sequence are reported to be able to selectively inhibit the function or expression of that particular nucleic acid by binding to or interfering with that nucleic acid, without disturbing the function or expression of other nucleic acid present in the cell. See, e.g., U.S. Pat. Nos. 4,469,863 and 4,511,713. The use of complementary nuclease-resistant nonionic oligonucleoside methylphosphonates which are taken up by mammalian cells to inhibit viral protein synthesis in certain contexts, including herpes simplex virus-1, is described in U.S. Pat. No. 4,757,055.
The inhibition of infection of cells by HTLV-III by administration of oligonucleotides complementary to highly conserved regions of the HTLV-III genome necessary for HTLV-III replication and/or expression is reported in U.S. Pat. No. 4,806,463. The oligonucleotides were said to affect viral replication and/or gene expression as assayed by reverse transcriptase activity (replication) and production of viral proteins p15 and p24 (gene expression).
Anti-sense oligonucleotides or phosphorothioate or other analogs complementary to a sequence of viral RNA theoretically may be employed to interrupt the transcription and translation of viral mRNA into protein. The anti-sense constructs can bind to viral mRNA and obstruct ribosomes from moving along the mRNA, thereby halting the translation of mRNA into protein. This process is called xe2x80x9ctranslation arrestxe2x80x9d or xe2x80x9cribosomal-hybridization arrest.xe2x80x9d Yarochan, et al., xe2x80x9cAIDS Therapiesxe2x80x9d, Scientific American, pages 110-119 (October, 1988).
However, in practice, the use of an anti-sense hybridizing sequence to obstruct the ribosome from reading along the mRNA is not generally useful in the coding portion of the target mRNA, since an anti-sense sequence targeted to the coding portion is often removed from hybridization with the target sequence during the course of translation, even where the binding constant is high. In contrast, an anti-sense sequence targeted to the 5xe2x80x2-untranslated position of a mRNA molecule may achieve translation arrest through a blocking type mechanism.
One approach to selective targeting of coding sequences is to rely on the ability of RNaseH to cleave duplexed RNA strands. In theory, by utilizing an anti-sense sequence which hybridizes to a target coding sequence on RNA, RNaseH cleavage of the target RNA could be achieved in this manner. However, in practice, cleavage by RNaseH requires that the strands in the target duplex sequence have 2xe2x80x2-deoxy sugar portions as well as charged (e.g., phosphodiester) backbone linkages. This means that uncharged-backbone anti-sense sequences such as methylphosphonate oligonucleosides (which are particularly useful because they are less subject to in vivo degradation) would not be expected to activate RNaseH activity against target RNA sequences. As a result, the increases in potency which can be achieved using modified oligonucleosides such as the methylphosphonates may not be realized (especially with respect to coding sequence targets) if RNaseH or translation arrest is relied upon to inhibit expression.
In an effort to increase the interference with protein synthesis of target genes and thereby increase potency, various agents can be bound to the anti-sense oligonucleotides which enhance the inactivation of the target nucleic acid. Such inactivating agents include alkylating agents, crosslinking agents and cleaving agents. These agents typically are capable of chemically modifying nucleic acid nonspecifically. By linking these agents to an anti-sense oligonucleotide, the target nucleic acid may in theory be modified or altered in specific locations.
Cleaving agents which have been covalently bound to oligonucleotides include, in particular, metal complexes such as EDTA-Fe(II), o-phenanthroline-Cu(II) and porphyrin-Fe(II). Uhlmann and Peyman, Antisense Oligonucleotides: A New Therapeutic Principle, Chem. Rev. 1990; 90:544-585. All of these cleaving agents require special conditions to perform the cleaving function. In each case, metal ion concentration must be carefully controlled to achieve cleavage. In some cases ancillary reagents not found in vivo, such as peroxide, are required, making such agents inappropriate for in vivo use. Baker has also recently reported experiments using Cu(II)-phenanthroline and other metal ion complexes for attempted hydrolysis of 5xe2x80x2-capped mono- and oligoribonucleotides. Baker, B. F., J. Am. Chem. Soc., 1993; 115:3378-3379.
Recently, a new class of non-site-specific nucleic acid cleaving agents has been investigated that do not require a metal ion free-radical mechanism to cleave the phosphodiester linkage. The design of these nucleic acid cleaving agents is intended to mimic the active site of naturally-occurring nucleases. Synthetic moieties modeled after nucleases such as staphylococcal nuclease have been reported to bind to phosphodiester linkages (Ariga and Ansilyn, J. Org. Chem. 1992; 57:417-419), as well as to accelerate both inter- and intramolecular phosphodiester cleavage (Jubian, et al., J. Am. Chem. Soc. 1992; 114:1120-1121). In another example, staphylococcal nuclease has been coupled to a synthetic oligonucleotide in an effort to achieve site-specific cleavage activity. Corey and Schultz, Science 1987; 238:1401-1403. However, this approach suffers from problems of immunogenicity, instability in biological fluids, and poor cellular uptake. Oligoamines, such as ethylenediamine, triethylenetetramine and pentaethylenehexamine, have been reported to accelerate the hydrolysis of RNA. Yoshiari, et al., Oligoamines as Simple and Efficient Catalysts for RNA Hydrolysis, J. Am. Chem. Soc. 1991; 113:5899-5901.
The present invention relates to the inhibition of expression of a target nucleic acid by contacting the target nucleic acid, particularly mRNA, with an oligonucleoside compound and causing selective degradation (cleavage) of the target nucleic acid. The present oligonucleoside compounds, referred to herein as xe2x80x9ccleavage compounds,xe2x80x9d are designed to target specific nucleic acid sequences by including an oligonucleoside sequence which is substantially complementary to the 5xe2x80x2-terminal portion, and particularly the region containing or adjacent to the 5xe2x80x2-cap structure of the target mRNA. Such cleavage compounds maximize the rate of 5xe2x80x2-cap structure cleavage, while retaining sequence specificity, by incorporating a Lewis acid or other electrophilic or electron-withdrawing moiety, preferably a metal ion complex, at a position in the oligonucleoside compound that is proximate to the 5xe2x80x2-cap structure of the target mRNA once the oligonucleoside compound hybridizes to the target. Since selective cleavage of the mRNA 5xe2x80x2-cap structure can prevent translation into encoded protein, the present compounds and methods prevent or reduce expression of the undesired protein encoded by the responsible gene. Such cleavage compounds may be expected to yield higher potencies than compounds associated with various other approaches to translation inhibition.
The compounds of the invention are believed to act primarily or even exclusively through a Lewis acid (electron-withdrawing) mechanism wherein the Lewis acid cleavage moiety of the cleavage compound withdraws electrons from the target phosphorus-oxygen center in the triphosphate 5xe2x80x2-cap structure, thereby facilitating direct nucleophilic attack by in situ water or hydroxide ion to effect hydrolytic cleavage. However, the invention is not limited to this particular proposed mode of activity, and other mechanisms may be applicable.
The cleavage compounds of the invention are preferably designed to form, in the course of cleavage, a hybridized duplex structure with a single-strand target RNA. However, the compounds may also be designed to form a triple-strand structure in the course of cleavage, as for example where two cleavage compounds act in tandem to form a triple-strand structure with a single-strand target RNA.
The cleavage compounds of the invention generally include a sequence of nucleosides that is chosen to be substantially complementary to a target region of the target nucleic acid strand, specifically the 5xe2x80x2-region of the target mRNA containing or adjacent to the 5xe2x80x2-cap structure, such that the cleavage compound is capable of hybridizing in a double-strand or triple-strand fashion to the 5xe2x80x2-region of target nucleic acid to effect cleavage of the cap structure. The xe2x80x9csubstantially complementaryxe2x80x9d portion is chosen so as to provide suitable target specificity and binding affinity of the cleavage compound. Oligonucleosides of the present invention are preferably between about 6 to 40 nucleosides in length, more preferably between 12 to 30 nucleosides. The length of a particular cleavage compound, the number of complementary bases in the compound, and the identity and location of the complementary bases may be adapted so that suitable target specificity and binding affinity will be achieved under the conditions in which the compound will be used. These conditions include, for example, the effective concentration of the cleavage compound inside the cell, the concentration and turnover rate of the target sequence, the desired level of reduction of concentration of the target sequence, the efficacy of cleavage, and the mode of cleavage (e.g., catalytic or non-catalytic).
The present oligonucleoside cleavage compounds preferably are modified to render them resistant to degradation by cellular nucleases or other enzymes that are present in vivo. This modification can be accomplished by methods known in the art, e.g., by incorporating one or more internal artificial internucleoside linkages, such as by modifying the phosphodiester linkage to include alternate or additional groups in conjunction with a phosphorus atom (e.g., by replacing one of the non-bridging phosphate oxygens in the linkage with sulfur or other atoms), and/or by blocking the 3xe2x80x2 end of the oligonucleoside with a capping structure. Likewise, the Lewis acid cleavage moiety portion can be conjugated to the cleavage compound using similar attachment chemistry, or using other techniques as described below. Multiple cleavage moieties can be conjugated to a single cleavage compound if desired, for example to increase the electrophilic cleavage-enhancing effect at the 5xe2x80x2-cap site.
Several advantages are provided by the cleavage compounds according to the present invention. The present compounds are target-mRNA-specific by virtue of their complementary oligonucleotide character, and therefore can be used against mRNA specific to a particular disease state. The cleavage agents are believed to facilitate hydrolysis by enhancing attack by an in situ nucleophilic species (especially water or hydroxide ion) on a phosphate ester bond of the triphosphate 5xe2x80x2-cap region of a target RNA sequence, and no free-radical mechanism utilizing metal ions or other compounds is required to implement the cleavage of the target RNA, making in vivo applications possible. Since the hydrolysis is performed by nucleophilic attack, rather than by hydroxyl free-radical attack as is common with some metal ion complexes, only the desired cleavage site should be hydrolyzed by the present compounds. Further, eliminating the creation of free radicals by the cleaving agent also allows in vivo use, as the present compounds are expected to be relatively harmless to non-targeted nucleic acid. The present compounds in some instances may also be catalytic in nature, permitting the administration of a relatively small amount of the present compounds for treatment.
Another feature of the present invention is the administration of the cleavage compounds described herein to treat diseases or other conditions characterized by the presence of undesired nucleic acid. The methods of the present invention are useful for inhibiting the expression of protein encoding genes. Administration of the present cleavage compounds can be accomplished by methods known in the art, such as systemic, topical or localized administration. Preferably, the present cleavage compounds are administered in an amount sufficient to prevent or reduce the normal translation of the target nucleic acid.
Other features and advantages of the present invention will be apparent upon review of the detailed description of the preferred embodiment, the drawings and the claims.